ABSTRACT The long-term goal of this project is to identify the mechanisms that regulate ?housekeeping? mitochondrial heme metabolism. Heme plays a central role in the redox reactions of life-essential processes such as mitochondrial respiration. My research program is particularly interested in 1. identifying proteins that regulate mitochondrial heme/porphyrin transport and 2. proteins that are part of the housekeeping mitochondrial homeostasis machinery that structurally or functionally interact with the core enzymes of the heme synthesis pathway. While the enzymes of the heme synthesis are well characterized and identical in all tissues, the regulatory mechanisms that couple heme synthesis to cellular requirements are very poorly understood. The importance of these regulatory mechanisms is underscored by the existence of disorders of heme synthesis and iron metabolism that are caused by dysregulation of proteins that are commonly associated with ?housekeeping? homeostatic processes or mitochondrial respiration. Although all tissues require heme, most of our studies on regulatory aspects of heme synthesis have focused on erythroid cells to the exclusion of understanding heme synthesis in other cell types. This project uses our knowledge of erythroid heme synthesis as a springboard to identify heme regulatory mechanisms that are required for housekeeping heme synthesis. Project 1 aims to identify proteins that are required for mitochondrial porphyrin transport in non-erythroid cells. Heme intermediates are photosensitive and cytotoxic, requiring mechanisms for cells to quickly and efficiently transport heme intermediates across cell membranes, to the next enzyme in the heme synthetic pathway. When efficient transport does not occur, cytotoxic porphyrins accumulate in cells, potentially causing porphyria and heme deficiency. While TMEM14C is essential for porphyrin transport in erythroid cells, TMEM14 proteins are not required for heme synthesis in non-erythroid cells. Using a combination of proteomics and whole genome sequencing/RNAseq analysis of TMEM14C suppressor mutants, we will identify novel mitochondrial proteins that regulate porphyrin transport in non-erythroid cells. The long-term goal of Project 2 is to understand the regulation of the heme synthesis complex by proteins that regulate housekeeping mitochondrial homeostasis. During this project period, we focus on the ubiquitous mitochondrial unfoldase, CLPX, which plays an essential role in regulating the activity and protein stability of heme synthesis enzymes. We show that the function of CLPX is highly tissue-specific, and propose to understand its role in the systemic regulation of heme metabolism in the setting of ?housekeeping? heme synthesis. These studies will provide essential datasets that will be invaluable to the mitochondrial protein unfoldase community, but will also be essential for determining how CLPX globally regulates mitochondrial homeostasis via tight regulation of heme.